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- EMDB-44204: Ubiquitin E2-Ub-E3 HECT tetrahedral transthiolation intermediate ... -

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Basic information

Entry
Database: EMDB / ID: EMD-44204
TitleUbiquitin E2-Ub-E3 HECT tetrahedral transthiolation intermediate mimic - state 5
Map dataFull map from the gold-standard refinement, globally sharpened using an B-factor of -40 A^2, used for model building and refinement.
Sample
  • Complex: Covalent E2-Ub-E3 HECT transthiolation intermediate mimic complex
    • Protein or peptide: Ubiquitin-conjugating enzyme E2 4
    • Protein or peptide: E3 ubiquitin-protein ligase pub2
  • Protein or peptide: Ubiquitin
  • Ligand: 4-aminobutanenitrile
KeywordsUBIQUITIN / E2 / E3 / HECT / NEDD4 / RSP5 / PUB2 / UBC4 / TRANSTHIOESTERIFICATION / THIOESTER / TRANSTHIOLATION / TETRAHEDRAL INTERMEDIATE / ADENYLATION / INHIBITOR / LIGASE / NUCLEUS / PHOSPHOPROTEIN / UBL CONJUGATION PATHWAY / UBL / ATP ATP-BINDING / AMP / NUCLEOTIDE-BINDING / ISOPEPTIDE BOND
Function / homology
Function and homology information


RHOQ GTPase cycle / RHOU GTPase cycle / Regulation of PTEN localization / Regulation of PTEN stability and activity / cytoplasm to vacuole targeting by the NVT pathway / cell cortex of cell tip / E3 ubiquitin ligases ubiquitinate target proteins / Peroxisomal protein import / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / nuclear SCF ubiquitin ligase complex ...RHOQ GTPase cycle / RHOU GTPase cycle / Regulation of PTEN localization / Regulation of PTEN stability and activity / cytoplasm to vacuole targeting by the NVT pathway / cell cortex of cell tip / E3 ubiquitin ligases ubiquitinate target proteins / Peroxisomal protein import / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / nuclear SCF ubiquitin ligase complex / Antigen processing: Ubiquitination & Proteasome degradation / SREBP signaling pathway / positive regulation of mitotic metaphase/anaphase transition / HECT-type E3 ubiquitin transferase / E2 ubiquitin-conjugating enzyme / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / cell division site / ubiquitin conjugating enzyme activity / protein K48-linked ubiquitination / ubiquitin ligase complex / modification-dependent protein catabolic process / protein polyubiquitination / protein tag activity / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / ribosome biogenesis / ubiquitin-dependent protein catabolic process / cytosolic large ribosomal subunit / cytoplasmic translation / protein ubiquitination / structural constituent of ribosome / ubiquitin protein ligase binding / nucleolus / ATP binding / membrane / nucleus / cytosol / cytoplasm
Similarity search - Function
E3 ubiquitin-protein ligase, SMURF1 type / HECT domain / HECT, E3 ligase catalytic domain / HECT-domain (ubiquitin-transferase) / HECT domain profile. / Domain Homologous to E6-AP Carboxyl Terminus with / Ubiquitin-conjugating enzyme, active site / Ubiquitin-conjugating (UBC) active site signature. / WW domain / WW/rsp5/WWP domain signature. ...E3 ubiquitin-protein ligase, SMURF1 type / HECT domain / HECT, E3 ligase catalytic domain / HECT-domain (ubiquitin-transferase) / HECT domain profile. / Domain Homologous to E6-AP Carboxyl Terminus with / Ubiquitin-conjugating enzyme, active site / Ubiquitin-conjugating (UBC) active site signature. / WW domain / WW/rsp5/WWP domain signature. / C2 domain / C2 domain profile. / WW domain superfamily / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme E2 / Ubiquitin-conjugating enzyme / Ubiquitin-conjugating (UBC) core domain profile. / WW/rsp5/WWP domain profile. / Domain with 2 conserved Trp (W) residues / WW domain / Ubiquitin-conjugating enzyme/RWD-like / Ribosomal L40e family / Ribosomal_L40e / Ribosomal protein L40e / Ribosomal protein L40e superfamily / C2 domain superfamily / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
Ubiquitin-ribosomal protein eL40B fusion protein / Ubiquitin-conjugating enzyme E2 4 / E3 ubiquitin-protein ligase pub2
Similarity search - Component
Biological speciesSchizosaccharomyces pombe 972h- (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.49 Å
AuthorsKochanczyk T / Lima CD
Funding support United States, 2 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118080 United States
CitationJournal: To be published
Title: Structural basis for transthiolation intermediates in the ubiquitin pathway
Authors: Kochanczyk T / Hann ZS / Lux MC / Reyes AMVD / Ji C / Tan DS / Lima CD
History
DepositionMar 22, 2024-
Header (metadata) releaseJun 5, 2024-
Map releaseJun 5, 2024-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44204.png

Downloads & links

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Map

FileDownload / File: emd_44204.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull map from the gold-standard refinement, globally sharpened using an B-factor of -40 A^2, used for model building and refinement.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 320 pix.
= 340.48 Å		1.06 Å/pix.
x 320 pix.
= 340.48 Å		1.06 Å/pix.
x 320 pix.
= 340.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.064 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.52
Minimum - Maximum-2.1152163 - 3.5582995
Average (Standard dev.)0.000210946 (±0.039712366)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 340.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Full map from the gold-standard refinement (unsharpened).

Fileemd_44204_additional_1.map
AnnotationFull map from the gold-standard refinement (unsharpened).
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 from the gold-standard refinement.

Fileemd_44204_half_map_1.map
AnnotationHalf map 1 from the gold-standard refinement.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 from the gold-standard refinement.

Fileemd_44204_half_map_2.map
AnnotationHalf map 2 from the gold-standard refinement.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Covalent E2-Ub-E3 HECT transthiolation intermediate mimic complex

EntireName: Covalent E2-Ub-E3 HECT transthiolation intermediate mimic complex
Components
  • Complex: Covalent E2-Ub-E3 HECT transthiolation intermediate mimic complex
    • Protein or peptide: Ubiquitin-conjugating enzyme E2 4
    • Protein or peptide: E3 ubiquitin-protein ligase pub2
  • Protein or peptide: Ubiquitin
  • Ligand: 4-aminobutanenitrile

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Supramolecule #1: Covalent E2-Ub-E3 HECT transthiolation intermediate mimic complex

SupramoleculeName: Covalent E2-Ub-E3 HECT transthiolation intermediate mimic complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1, #3
Source (natural)Organism: Schizosaccharomyces pombe 972h- (yeast) / Strain: 972 / ATCC 24843

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Macromolecule #1: Ubiquitin-conjugating enzyme E2 4

MacromoleculeName: Ubiquitin-conjugating enzyme E2 4 / type: protein_or_peptide / ID: 1
Details: C-terminal GGLVPR is a residual artifact after thrombin cleavage of affinity tag
Number of copies: 1 / Enantiomer: LEVO / EC number: E2 ubiquitin-conjugating enzyme
Source (natural)Organism: Schizosaccharomyces pombe 972h- (yeast) / Strain: 972 / ATCC 24843
Molecular weightTheoretical: 17.043336 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MALKRINREL ADLGKDPPSS SSAGPVGDDL FHWQATIMGP ADSPYAGGVF FLSIHFPTDY PFKPPKVNFT TRIYHPNINS NGSICLDIL RDQWSPALTI SKVLLSISSL LTDPNPDDPL VPEIAHVYKT DRSRYELSAR EWTRKYAIGG LVPR

UniProtKB: Ubiquitin-conjugating enzyme E2 4

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Macromolecule #2: Ubiquitin

MacromoleculeName: Ubiquitin / type: protein_or_peptide / ID: 2
Details: N-terminal GSGG is a residual artifact after TEV protease cleavage of affinity tag
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Schizosaccharomyces pombe 972h- (yeast) / Strain: 972 / ATCC 24843
Molecular weightTheoretical: 8.769948 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
GSGGMQIFVK TLTGKTITLE VESSDTIDNV KSKIQDKEGI PPDQQRLIFA GKQLEDGRTL SDYNIQKEST LHLVLRLRG

UniProtKB: Ubiquitin-ribosomal protein eL40B fusion protein

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Macromolecule #3: E3 ubiquitin-protein ligase pub2

MacromoleculeName: E3 ubiquitin-protein ligase pub2 / type: protein_or_peptide / ID: 3
Details: N-terminal SHM is a residual artifact after cleaving the affinity tag
Number of copies: 1 / Enantiomer: LEVO / EC number: HECT-type E3 ubiquitin transferase
Source (natural)Organism: Schizosaccharomyces pombe 972h- (yeast) / Strain: 972 / ATCC 24843
Molecular weightTheoretical: 44.231828 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SHMDEYQRKI AYMYDRPEMA VNDAQLQLKV SRATTFEDAY DIISKLSVSD MKKKLLIRFR NEDGLDYGGV SREFFYILSH AIFNPGYSL FEYATDDNYG LQISPLSSVN PDFRSYFRFV GRVMGLAIYH RRYLDVQFVL PFYKRILQKP LCLEDVKDVD E VYYESLKW ...String:
SHMDEYQRKI AYMYDRPEMA VNDAQLQLKV SRATTFEDAY DIISKLSVSD MKKKLLIRFR NEDGLDYGGV SREFFYILSH AIFNPGYSL FEYATDDNYG LQISPLSSVN PDFRSYFRFV GRVMGLAIYH RRYLDVQFVL PFYKRILQKP LCLEDVKDVD E VYYESLKW IKNNDVDESL CLNFSVEENR FGESVTVDLI PNGRNIAVNN QNKMNYLKAL TEHKLVTSTE EQFNALKGGL NE LIPDSVL QIFNENELDT LLNGKRDIDV QDWKRFTDYR SYTETDDIVI WFWELLSEWS PEKKAKLLQF ATGTSRLPLS GFK DMHGSD GPRKFTIEKV GHISQLPKAH TCFNRLDIPP YNSKEELEQK LTIAIQETAG FGTE

UniProtKB: E3 ubiquitin-protein ligase pub2

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Macromolecule #4: 4-aminobutanenitrile

MacromoleculeName: 4-aminobutanenitrile / type: ligand / ID: 4 / Number of copies: 1 / Formula: A1AIV
Molecular weightTheoretical: 84.12 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration4.5 mg/mL
BufferpH: 7.2
Component:
ConcentrationFormulaName
20.0 mMC4H12NO3Tris HCl buffer
100.0 mMNaClSodium chlorideSodium chloride
1.0 mg/mlC32H59N2O8SCHAPSOCHAPS detergent

Details: 20 mM Tris-HCl, 100 mM NaCl, 0.1% CHAPSO
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: SUPER-RESOLUTION / Average exposure time: 4.0 sec. / Average electron dose: 72.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Ab initio model from cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.49 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 79978
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9b59:
Ubiquitin E2-Ub-E3 HECT tetrahedral transthiolation intermediate mimic - state 5

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